For the decade prior to the decade hereof, glucose/fructose mixtures manufactured from corn starch constituted an ever-growing segment of the U.S. sugar industry. This segment of the sugar industry has grown to such size as to demand substantial quantities of the starch converting enzymes which generate the glucose/fructose mixtures. In particular, industrial usage of thermally stable alpha-amylase to hydrolyze the corn starch has grown into a market worth several million dollars per annum to suppliers of the enzyme.
Heretofore, the alpha-amylase from Bacillus licheniformis, (e.g., TERMAMYL.RTM.) was the thermally stable enzyme of choice. As of the date hereof, however, the art has been offered an opportunity to shift to the thermally stable alpha-amylase elaborated by Bacillus stearothermophilus (e.g., THERMOLASE.TM.). For background on this alpha-amylase, reference is made to Pat. Nos. 2,695,863 and 4,284,722.
The B. stearothermophilus enzyme has certain advantages over the Bacillus licheniformis enzyme, notably, a higher specific activity, a lower pH optimum, and a modest improvement in dextrose yield. Unfortunately, liquefying starch with the B. stearothermophilus enzyme causes appearance of significant levels of sediment in the glucose syrup unless a high dosage level of the B. stearothermophilus enzyme is employed. Costs of removing the sediment from the syrup largely negate the advantages offered by this enzyme.
Analysis of the sediment generated in the syrup by use of the B. stearothermophilus alpha-amylase at normal dosage levels indicates that the sediment constitutes a polysaccharide, which polysaccharide may be complexed with lipid and protein. Apparently some component in the starch is poorly hydrolyzed by this alpha-amylase, since this sediment problem can be resolved by increasing dosage of the alpha-amylase substantially. The possibility exists, therefore, that treatment of either the starch or the dextrin with small amounts of some enzyme effective against the sediment forming fraction, might resolve the sediment problem.
Liquefying starch at pH 6.0-6.5 with normal dosage levels of the Bacillus licheniformis enzyme does not generate undue levels of sediment. However, when liquefying with the Bacillus licheniformis enzyme at pH 5.5-6.0 excessive sediment is also generated (and otherwise relatively poor liquefaction results).
The below-tabulated test results from laboratory scale comparative studies wherein the liquefaction was simulative of industrial usage circumstances and standard saccharification practices were followed illustrates the degree to which sediment appears at different liquefaction pH and enzyme dosage.
______________________________________ Sediment Volume (% vol/vol) Liquefaction pH Liquefaction Enzyme 5.0 5.5 5.8 6.0 ______________________________________ TERMAMYL .RTM. 2.5*, ** 2.0* 1.5 85 NU/g DS THERMOLASE .TM. 50 10 7 25 50 NU/g DS THERMOLASE .TM. 5 50 NU/g DS THERMOLASE .TM. 2 100 NU/g DS ______________________________________ *Accompanied by poor liquefaction results **At pH 5.5 twice the usual Ca.sup.++ level was employed to stabilize th TERMAMYL .RTM.-
The activity standard NU (which is an abbreviation of NOVO alpha-amylase unit) is the amount of enzyme which breaks down 5.26 mg of dissolved starch per hour at 37.degree. C., pH 5.6 and 0.0043M of Ca.sup.++ over a 7-20 minute reaction time. The analytical method AF-9 is available on request to NOVO INDUSTRI A/S, DK-2880 Bagsvaerd, Denmark. At the starch liquefaction operating range of 90.degree.-110.degree. C., the THERMOLASE.TM. has been found to be more active than the TERMAMYL.RTM. by a factor of about 1.7. The test study results tabulated above which compared TERMAMYL.RTM. and THERMOLASE.TM. at starch liquefaction temperatures was an equal activity level study i.e., 50 NU/g DS of THERMOLASE.TM. is as effective as 85 NU/g DS of TERMAMYL.RTM. at the conditions of use.
Surprisingly, liquefying starch at pH 5.5-6.0 with a mixture of the Bacillus licheniformis alpha-amylase and the B. stearothermophilus alpha-amylase eliminated the sediment problem without harm to liquefaction results.